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Molecuar basis of the development of GABAergic neuron using GFP knock-in mouse

GFP敲入小鼠GABA能神经元发育的分子基础

基本信息

批准号：
12680789
负责人：
YANAGAWA Yuchio
金额：
$ 2.37万
依托单位：
Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

In order to elucidate the molecular mechanisms of the development of GABAergic neurons, we labeled the GABAergic neurons with a reporter protein. First, we used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DMA fragment of the glutamate decarboxylase (GAD) 65 gene fused to the bacterial /acZ reporter gene. Transgenic mice showed high β-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of the transgene expression in olfactory bulb, cerebellar cortex and spinal cord, a similar expression pattern to that of endogenous GAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high GAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DMA fragment of the GAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions, but that additional regulatory elements are necessary to obtain fully correct expression. Second, we generated the GAD67-GFP mutant mice using knock-in method. In the GAD67-GFP mice, green fluorescence was observed in the GABAergic neurons without fixation. We utilized GAD67-GFP mice to observe distribution and migration of GABAergic neurons in the forebrain of the embryonic mouse. GFPpositve GABAergic neurons were found in the ganglionic eminences and the septum from Ell.5 and onwards. GABAergic neurons were also observed in the marginal zone and intermediate zone of the cortex. Using time-lapse imaging we observed that a large population of telencephalic GABAergic neurons migrate from the basal forebrain towards the cortex. These data suggest that GAD67-GFP mice will be useful for elucidating the development of GABAergic neurons.

为了阐明GABA能神经元发育的分子机制，我们用报告蛋白标记GABA能神经元。首先，我们使用转基因小鼠检测了9.2kb的谷氨酸脱羧酶(GAD)65基因启动子-近端DMA片段与细菌/acZ报告基因融合后的表达模式。转基因小鼠在脑和睾丸中表现出较高的β-半乳糖苷酶活性。它们还显示出转基因在嗅球、小脑皮质和脊髓中表达的特征模式，与内源性GAD65的表达模式相似。然而，在腹侧丘脑和下丘脑中没有观察到转基因表达，其中GAD65基因表达水平较高。这些结果表明，GAD65基因的9.2kb的DMA片段与其组织特异性表达和特定脑区GABA能神经元的靶向表达有关，但额外的调控元件是获得完全正确表达所必需的。其次，我们用敲入法获得了GAD67-GFP突变小鼠。在GAD67-GFP小鼠的GABA能神经元中观察到无固定的绿色荧光。我们利用GAD67-GFP小鼠观察了GABA能神经元在胚胎小鼠前脑中的分布和迁移。GFP阳性的GABA能神经元分布于神经节隆起和隔区。大脑皮质边缘带和中间带也可见GABA能神经元。利用延时成像，我们观察到大量的端脑GABA能神经元从基底前脑向皮质迁移。这些数据表明，GAD67-GFP小鼠将有助于阐明GABA能神经元的发育。